FANCD2 interaction with mismatch repair proteins and MCM2-7

描述(由申请人提供)：范可尼贫血(FA)在细胞水平上的表型以基因组不稳定和对DNA损伤高度敏感为特征。FA蛋白参与与FA途径并列的复合体，这些复合体与其他更明确的参与细胞对DNA损伤的反应的途径并列，如ATR、BRCA1和RAD51。最近的工作将FA生物学的重点和这些蛋白质的正常功能转移到同源重组途径上。然而，由于FA蛋白几乎不包含已知的功能基序，在正常的FA生物化学或FA蛋白在造血和白血病中的作用方式方面仍有许多有待确定的地方。深入了解FA的生化途径很重要，因为这将导致基因组不稳定发生的机制，这是癌症的基本原因。这一认识使癌症预防的干预措施成为可能，也使操纵基因组不稳定性用于癌症治疗成为可能。我的实验室的长期目标是以FA为模型，确定基因组不稳定的机制，以及了解FA蛋白的正常功能。这项研究的中心假设是FANCD2作为信号转导蛋白与下游效应器相互作用，如MSH2、错配修复蛋白和MCM2-7复合体，这对复制机制的起始前加载至关重要。我们的工作表明，这两个过程都依赖于上游的磷酸化事件。为了检验我们的中心假设并实现这一应用，我们将追求以下具体目标：目标1：确定FANCD2-MSH2相互作用的功能后果目标1的工作假设是FANCD2-MSH2相互作用对于正常的DNA损伤反应是重要的。我们的数据表明，MSH2基因敲除不仅会导致DNA损伤超敏反应，而且FANCD2和MSH2基因的双重敲除也会相反地导致细胞耐药。目的#2：确定FANCD2-MLH1相互作用的功能结果目的#2的工作假设是FANCD2-MLH1相互作用对正常的DNA损伤反应是重要的。我们发现MLH1的缺失并不影响FANCD2的单价基化，但确实阻止了信号向染色质的传播以及随后对DNA交联剂的敏感性增加。目的#3：证明FANCD2-MCM2-7相互作用的功能结果目的#2的工作假设是FANCD2-MCM2-7相互作用通过对S期DNA损伤的反应来调节复制复合体在起点的组装。我们的数据表明，与MCM亚基的一个子集的结合导致了复制复合体组装的抑制。了解FA途径将进一步阐述关键的修复途径，并为靶向癌症治疗的途径提供洞察力。 公共卫生相关性：Fanconi贫血(FA)是一种癌症易感性的遗传病，其解剖导致了对DNA损伤反应和同源重组修复的研究。我们发现FA途径中的中心蛋白FANCD2通过MSH2和MLH1以及复制的解旋酶MCM2-7与错配修复途径相互作用。我们假设FANCD2通过这些蛋白调节修复和复制的行为。对这些途径的研究将使我们不仅能够了解DNA修复和反应的基本机制，而且还将揭示调节癌症机制和癌症治疗反应的途径。

DESCRIPTION (provided by applicant): The Fanconi anemia (FA) phenotype on a cellular level is marked by genomic instability and hypersensitivity to DNA damage. FA proteins participate in complexes that juxtapose the FA pathway with other more defined pathways involved in cellular response to DNA damage, such as ATR, BRCA1, and RAD51. Recent work has served to shift focus of FA biology and the normal functions of these proteins onto homologous recombination pathways. However, much remains to be ascertained in normal FA biochemistry or about the manner in which FA proteins contribute to hematopoiesis and leukemia, as the FA proteins contain few known functional motifs. Insight into the biochemical pathway of FA is important because this would lead to a mechanism whereby genomic instability occurs, which is a basic cause of cancer. This understanding makes possible interventions for cancer prevention as well as manipulation of genomic instability for cancer therapy. The long-term goal of my laboratory is to define mechanisms of genomic instability, using FA as a model, as well as to understand the normal function of FA proteins. The central hypothesis for the proposed research is that FANCD2 acts as a signal transduction protein to interact with downstream effectors such as the MSH2, the mismatch repair protein, and the MCM2-7 complex, which is critical for pre-origin loading of the replication machinery. Our work suggests that both these processes depend on upstream phosphorylation events. To test our central hypothesis and carry out this application, we will pursue the following specific aims: Aim #1: Determine the functional consequence of FANCD2-MSH2 interaction The working hypothesis for Aim #1 is that FANCD2-MSH2 interaction is important for the normal DNA damage response. Our data have shown that not only does MSH2 knockdown result in DNA damage hypersensitivity but also double knockdown of both FANCD2 and MSH2 contradictorily results in cellular resistance. Aim #2: Determine the functional consequence of FANCD2-MLH1 interaction The working hypothesis for Aim #2 is that FANCD2-MLH1 interaction is important for the normal DNA damage response. We show that MLH1 loss does not affect FANCD2 monoubiquitylation but does prevent the propagation of signal to chromatin and subsequent increased sensitivity to DNA crosslinkers. Aim #3: Demonstrate the functional consequence of FANCD2-MCM2-7 interaction The working hypothesis for Aim #2 is that FANCD2-MCM2-7 interaction regulates the assembly of the replication complex at the origin by responding to DNA damage during S phase. Our data indicate that binding to a subset of the MCM subunits results in inhibition of replication complex assembly. Understanding the FA pathway will further elaborate a key repair pathway as well as lend insight into avenues of targeting cancer therapy. PUBLIC HEALTH RELEVANCE: Fanconi anemia (FA) is a genetic disease of cancer susceptibility whose dissection has led to the study of the DNA damage response and homologous recombinatorial repair. We have found that the central protein FANCD2 in the FA pathway interacts with the mismatch repair pathway via MSH2 and MLH1 as well the replicative helicase MCM2-7. We hypothesize that FANCD2 regulates the action of repair and replication through these proteins. Study of these pathways will allow us to not only understand basic mechanisms of DNA repair and response but also will uncover avenues whereby mechanisms of cancer and response to cancer therapy may be modulated.